Dynamic Range of Mass Accuracy in LTQ Orbitrap Hybrid Mass Spectrometer
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Tandem Mass Spectrometry (MS–MS)
Advanced Analytical Chemistry Lecture 22 Chem 4631 Tandem Mass Spectrometry (MS–MS) Tandem mass spectrometry (MS–MS) is a term which covers a number of techniques where one stage of mass spectrometry (not necessarily the first) is used to isolate an ion of interest and a second stage is then used to probe the relationship of this ion with others from which it may have been generated or which it may generate on decomposition. Chem 5570 Tandem Mass Spectrometry (MS–MS) Chem 5570 The two analyzers (MS-MS) can be separated by a collision cell (can be another MS) into which an inert gas (e.g. argon, xenon) is admitted to collide with the selected sample ions and bring about their fragmentation. Tandem MS have the ability to perform multiple steps on a single sample. The MS selects a specific ion, fragment the ion, and generate another mass spec – able to repeat the cycle several times. Chem 5570 The analyzers can be of the same or of different types, the most common combinations being: quadrupole - quadrupole magnetic sector - quadrupole magnetic sector - magnetic sector quadrupole - time-of-flight Fragmentation experiments can also be performed on certain single analyzer mass spectrometers such as ion trap and time-of-flight instruments, the latter type using a post-source decay experiment to effect the fragmentation of sample ions. Chem 5570 Tandem Mass Spectrometry (MS–MS) TIC - Total ion current or total ion chromatogram The TIC represents the sum of all signal intensities of a single scan spectrum. The TIC is usually calculated by the data system of the mass spectrometer and plotted against time or scan number to give a measure for evaporation/ionization of a sample over the duration of the whole measurement. -
Front-End Methods for Enhancing the Analytical Power of Mass Spectrometry
FRONT-END METHODS FOR ENHANCING THE ANALYTICAL POWER OF MASS SPECTROMETRY PETER PAUL LIUNI A DISSERTAITON SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN CHEMISTRY YORK UNIVERSITY TORONTO, ONTARIO December 2015 © Peter Paul Liuni, 2015 Abstract The analytical power and versatility of mass spectrometry can be enhanced by adding ‘front-end’ devices, which provide additional functionality before, during or immediately after ElectroSpray Ionization (ESI). Such devices can include Ion mobility spectrometry (IMS) and Time-Resolved ElectroSpray Ionization (TRESI) which provide enhanced analysis of illicit compounds, protein folding, enzyme kinetics, and catalysis-linked dynamics. With respect to IMS, this work describes implementation of a hybrid Trace Compound Detector (TCD) system that combines IMS and MS to allow for rapid front- end mobility separation, followed by characterization and identification of analytical markers of seized opium by mass spectrometry. Ultimately, this device provides an avenue for rapid prosecution based on simultaneous detection and unambiguous identification of illicit drugs. TRESI is used to extend Mass Spectrometry (MS) to millisecond-timescale reaction studies. In the first instance, we combine TRESI with Travelling Wave Ion Mobility Spectrometry (TWIMS) to compare equilibrium and kinetic unfolding intermediates of cytochrome c, showing a high degree of correlation between all species populated under these substantially different regimes. We then combine TRESI with Hydrogen Deuterium Exchange (TRESI-HDX) to elucidate the relationship between structural fluctuations (conformational dynamics) of enzymes and their catalytic activity. The results of this work include a new model for catalysis-linked dynamics, in which the nature of the conformational landscape explored by an enzyme is independent of catalysis, but the rate at which the landscape is explored is enhanced for catalytically active species. -
Novel Quadrupole Time-Of-Flight Mass Spectrometry for Shotgun Proteomics
DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER FAKULTÄT FÜR CHEMIE UND PHARMAZIE DER LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN Novel quadrupole time-of-flight mass spectrometry for shotgun proteomics von Scarlet Svenja Anna-Maria Beck aus Tettnang 2016 ii Erklärung Diese Dissertation wurde im Sinne von §7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Matthias Mann betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 25.04.2017 …………………………………………………………………………………………Scarlet Beck Dissertation eingereicht am 23.09.2016 1. Gutachter: Prof. Dr. Matthias Mann 2. Gutachter: Prof. Dr. Jürgen Cox Mündliche Prüfung am 04.11.2016 iii iv ABSTRACT Mass spectrometry (MS)-based proteomics has become a powerful technology for the identification and quantification of thousands of proteins. However, the coverage of complete proteomes is still very challenging due to the high sample complexity and the difference in protein concentrations. In data-dependent shotgun proteomics several peptides elute simultaneously from the column and are isolated by the quadrupole and fragmented by the collision cell one at a time. This method has two major disadvantages. On the one hand, a large number of eluting peptides cannot be targeted since the sequencing speeds of current instruments are too slow and on the other hand, peptides that only differ slightly in mass and elute together are co-isolated and co-fragmented, resulting in chimeric MS2 spectra. Therefore an urgent need for further developments and improvements of mass spectrometers remains. The aim of this thesis was to co-develop, evaluate and improve novel quadrupole time-of-flight (QTOF) mass spectrometers. In my first project I have described the developments and improvements of the hardware of the high-resolution QTOF mass spectrometer, the impact II, and have shown that this instrument can be used for very deep coverage of diverse proteomes as well as for accurate and reproducible quantification. -
Peptide and Protein Quantification Using Itraq with Electron Transfer Dissociation
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Peptide and Protein Quantification Using iTRAQ with Electron Transfer Dissociation Doug Phanstiel,a Yi Zhang,c Jarrod A. Marto,c,d and Joshua J. a,bCoon a Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA b Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA c Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts, USA d Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA Electron transfer dissociation (ETD) has become increasingly used in proteomic analyses due to its complementarity to collision-activated dissociation (CAD) and its ability to sequence peptides with post-translation modifications (PTMs). It was previously unknown, however, whether ETD would be compatible with a commonly employed quantification technique, isobaric tags for relative and absolute quantification (iTRAQ), since the fragmentation mechanisms and pathways of ETD differ significantly from CAD. We demonstrate here that ETD of iTRAQ labeled peptides producesc- and z˙ -type fragment ions as well as reporter ions that are unique from those produced by CAD. Exact molecular formulas of product ions were determined by ETD fragmentation of iTRAQ-labeled synthetic peptides followed by high mass accuracy orbitrap mass analysis. These experiments revealed that ETD cleavage␣ of the N–C bond of the iTRAQ tag results in fragment ions that could be used for quantification. Synthetic peptide work demonstrates that these fragment ions provide up to three channels of quantification and that the quality is similar to that provided by beam-type CAD. -
Implementation of Electron-Transfer Dissociation on a Hybrid Linear Ion Trap-Orbitrap Mass Spectrometer
Anal. Chem. 2007, 79, 3525-3534 Accelerated Articles Implementation of Electron-Transfer Dissociation on a Hybrid Linear Ion Trap-Orbitrap Mass Spectrometer Graeme C. McAlister,† Doug Phanstiel,† David M. Good,† W. Travis Berggren,‡ and Joshua J. Coon*,†,§ Departments of Chemistry and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, and WiCell Research Institute, Madison, Wisconsin 53706 We describe the adaptation of a hybrid quadrupole linear electron-transfer dissociation (ETD) has generated considerable ion trap-orbitrap mass spectrometer to accommodate interest in the field of proteomic research.1-3 The utility of the electron-transfer ion/ion reactions (ETD) for peptide and technique to localize post-translational modifications (PTMs), its protein characterization. The method utilizes pulsed, dual relative indifference to amino acid composition or order, and electrospray ion sources and requires minimal instrument capacity to randomly dissociate large peptide and even whole modification. Switching between cation and reagent anion protein cations on a chromatographic time scale make it the injection schemes is automated and accomplished within perfect complement to conventional collision-activated methodol- a few hundred milliseconds. Ion/ion reactions are con- ogy (CAD).4-8 Still, because they are generated within the context ducted within the linear ion trap, after which the c- and of a radio frequency (rf) ion trap, ETD-type product ions are almost z-type product ions are passed to the orbitrap for high- exclusively mass analyzed with low m/z resolution and accuracy resolution m/z analysis. With this arrangement, mass (i.e., that typically achieved with ion trap devices). Doubtless ion accuracies are typically measured to within 2 ppm at a trap MS systems offer a splendid format for conducting ion/ion resolving power of 60 000. -
Utilizing a Hybrid Mass Spectrometer to Enable Fundamental Protein Characterization: Intact Mass Analysis and Top-Down Fragmentation with the LTQ Orbitrap MS
Application Note: 498 Utilizing a Hybrid Mass Spectrometer to Enable Fundamental Protein Characterization: Intact Mass Analysis and Top-Down Fragmentation with the LTQ Orbitrap MS Tonya Pekar Second, Vlad Zabrouskov, Thermo Fisher Scientific, San Jose, CA, USA Alexander Makarov, Thermo Fisher Scientific, Bremen, Germany Introduction Experimental Key Words A fundamental stage in protein characterization is to Protein standards, including bovine carbonic anhydrase, • LTQ Orbitrap Velos determine and verify the intact state of the macromolecule. yeast enolase, bovine transferrin and human monoclonal This is often accomplished through the use of mass IgG, were purchased from Sigma-Aldrich. For direct • LTQ Orbitrap XL spectrometry (MS) to first detect and measure the molecular infusion, proteins in solution were purified by either a • Applied mass. Beyond confirmation of intact mass, the next objective Thermo Scientific Vivaspin centrifugal spin column or a Fragmentation is the verification of its primary structure, the amino acid size-exclusion column (GE Healthcare), employing at least Techniques sequence of the protein. Traditionally, a map of the two rounds of buffer exchange into 10 mM ammonium macromolecule is reconstructed from matching masses of acetate. Protein solutions were at a concentration of least • Electron Transfer peptide fragments produced through external enzymatic 1 mg/mL prior to clean-up. Samples were diluted into Dissociation ETD digestion of the protein to masses calculated from an in 50:50:0.1 acetonitrile:water:formic acid prior to infusion silico • Top-Down digest of the target protein sequence. A more direct into the mass spectrometer. Instrument parameters were approach involves top-down MS/MS of the intact protein altered during infusion of protein solutions to optimize the Proteomics molecular ion. -
Orbitrap Elite Hybrid MS Provides the Ultimate Level of Versatility and Performance for Your Research
mass spectrometry Product Specifications Combining the next generation Thermo Scientific Velos Pro ion trap with the new high-field Orbitrap technology, the Thermo Scientific Orbitrap Elite hybrid MS provides the ultimate level of versatility and performance for your research. Thermo Scientific Orbitrap Elite High-Field Orbitrap Hybrid Mass Spectrometer • Novel high-field Orbitrap analyzer The ultrahigh resolution provides • Resolving power > 240,000 FWHM certainty in analytical results by enabling • Advanced signal processing molecular weight determination for intact • Multiple fragmentation techniques: CID, proteins and in-depth analysis of isobaric HCD and optionally ETD species. The unsurpassed resolution of the • Parallel MS and MSn analysis system is especially useful when dealing • Velos Pro ion trap generation II ion optics with complex and low abundance samples, with neutral blocking technology increase in applications such as proteomics, metabo- robustness lomics and lipidomics. The Orbitrap Elite hybrid MS can The Thermo Scientific Orbitrap Elite MS is perform a wide variety of experiments from the industry-leading high performance in-depth discovery based experiments to hybrid mass spectrometer combining the comprehensive qual/quan experiments. The novel high-field Orbitrap™ analyzer with availability of multiple fragmentation tech- the latest dual-pressure linear ion trap, the niques (CID, HCD and optional ETD) offers Thermo Scientific Velos Pro. a new level of versatility and performance The high-field Orbitrap analyzer -
Discovery and Quantitation of Protein Modifications Using Targeted Mass
Discovery and Quantitation of Protein Modifications using Targeted Mass Spectrometry Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jia You, M.E. Graduate Program in Chemistry The Ohio State University 2012 Dissertation Committee: Dr. Michael A. Freitas, Advisor Dr. Dehua Pei, Co-Advisor Dr. Anne Co Copyright by Jia You 2012 Abstract In this dissertation, efforts were focused on the development of targeted proteomic assays to elucidate differences in protein profiles present in diseases and their correlation with other molecular markers (proteins or microRNA). In Chapter 2, a high-sensitivity TFA-free LC-MS method is described. The analysis of proteins by reversed-phase liquid chromatography (RPLC) commonly involves the use of TFA as an ion-pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC-MS, TFA-free methods for protein separation optimized. Protein standards and histones were used to evaluate TFA-free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C8 columns with the ion-pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients. ii In chapter 3, a targeted mass spectrometry approach was used to examine nitration and nitrosylation of tyrosine residues on tropomyosin. -
Comparison of Collisional and Electron-Based Dissociation Modes For
Manuscript Click here to view linked References 1 Middle-down tandem MS of glycopeptides 1 2 3 4 5 6 Comparison of collisional and electron-based dissociation modes for middle-down analysis of multiply 7 8 glycosylated peptides 9 1 2 3 1,2 #1 #1,3 10 Kshitij Khatri *, Yi Pu *, Joshua A. Klein , Catherine E. Costello , Cheng Lin , Joseph Zaia 11 12 13 14 1Dept. of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of 15 Medicine 16 17 2 18 Dept. of Chemistry, Boston University 19 3 20 Program in Bioinformatics, Boston University 21 22 23 24 25 26 *Contributed equally 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 #Address for correspondence 42 43 44 Joseph Zaia/Cheng Lin 45 46 Boston University Medical Campus 47 48 670 Albany St., Rm. 509 49 50 Boston, MA 02118 51 52 (v) 1-617-638-6762 53 54 (f) 1-617-638-6761 55 56 57 (e) [email protected]; [email protected] 58 59 60 61 62 63 64 65 1 Middle-down tandem MS of glycopeptides 2 2 3 4 Abstract 5 6 Analysis of singly glycosylated peptides has evolved to a point where large-scale LC-MS analyses can be 7 8 9 performed at almost the same scale as proteomics experiments. While collisionally activated dissociation 10 11 (CAD) remains the mainstay of bottom-up analyses, it performs poorly for the middle-down analysis of 12 13 multiply glycosylated peptides. With improvements in instrumentation, electron-activated dissociation 14 15 (ExD) modes are becoming increasingly prevalent for proteomics experiments and for the analysis of 16 17 18 fragile modifications such as glycosylation. -
Second-Generation Electron Transfer Dissociation (ETD) on the Thermo
Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientifi c Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen,1 Lee Earley,1 Jean-Jacques Dunyach,1 John E.P. Syka,1 Philip D. Compton,2 Dina L. Bai,3 Jefferey Shabanowitz,3 and Donald F. Hunt3 1Thermo Fisher Scientifi c, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientific Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Philip D. Compton2, Dina L. Bai3, Jefferey Shabanowitz3, and Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA TABLE 1. Calculated charge state dependent reaction times based on a Verification of the Calibration Unique ETD Capabilities Overview Results saturated reaction rate coefficient of 58.2 sec–1 for charge state 3+, based on 5% The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer of the precursor remaining after reaction. Purpose: Improve ETD robustness, functionality, and speed on the Thermo Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is from bovine erythrocytes. Spectra of the 12+ charge state at 714.7 m/z were obtained allows for a parallel ITMS2 acquisition mode, enabling ITMS2 CID and HCD spectral Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer optimized and that the maximum duty cycle for ETD can be accomplished. -
Mass Spectrometry: Forming Ions, to Identifying Proteins and Their Modifications
Mass spectrometry: forming ions, to identifying proteins and their modifications Stephen Barnes, PhD 4-7117 [email protected] S Barnes-UAB 1/20/06 Introduction to mass spectrometry • Class 1 - Biology and mass spectrometry – Why is mass spectrometry so important? – Short history of mass spectrometry – Ionization and measurement of ions • Class 2 - The mass spectrum – What is a mass spectrum? – Interpreting ESI and MALDI-TOF spectra – Combining peptide separation with mass spectrometry – MALDI mass fingerprinting – Tandem mass spectrometry and peptide sequencing S Barnes-UAB 1/20/06 1 Goals of research on proteins • To know which proteins are expressed in each cell, preferably one cell at a time • Major analytical challenges – Sensitivity - no PCR reaction for proteins – Larger number of protein forms than open reading frames – Huge dynamic range (109) – Spatial and time-dependent issues S Barnes-UAB 1/20/06 Changes at the protein level • To know how proteins are modified, information that cannot necessarily be deduced from the nucleotide sequence of individual genes. • Modification may take the form of – specific deletions (leader sequences), – enzymatically induced additions and subsequent deletions (e.g., phosphorylation and glycosylation), – intended chemical changes (e.g., alkylation of sulfhydryl groups), – and unwanted chemical changes (e.g., oxidation of sulfhydryl groups, nitration, etc.). S Barnes-UAB 1/20/06 2 Proteins once you have them Protein structure and protein-protein interaction – to determine how proteins assemble in solution – how they interact with each other – Transient structural and chemical changes that are part of enzyme catalysis, receptor activation and transporters S Barnes-UAB 1/20/06 So, what you need to know about mass spec • Substances have to be ionized to be detected. -
N- and C-Terminal Sequencing of Proteins Using Top-Down Electron Transfer Dissociation Mass Spectrometry
Application Note: 484 N- and C-terminal Sequencing of Proteins Using Top-down Electron Transfer Dissociation Mass Spectrometry Zhiqi Hao, Lihua Jiang, Iman Mohtashemi, Andreas FR Hühmer, Thermo Fisher Scientific, San Jose, CA, USA Yuehe Ding, Mengqiu Dong, National Institute of Biological Sciences, Beijing, China Introduction proteins is presented. The high-resolution, high-mass- Key Words N-terminal sequencing is widely accepted as a reliable tool accuracy capabilities of Orbitrap ETD hybrid mass spectrometry coupled with multiple fragmentation techniques • Orbitrap for protein characterization throughout all stages of drug discovery and biopharmaceutical manufacturing. The two enabled complete characterization of N- and C-termini of • C-terminal major direct methods of protein N-terminal sequencing a truncated protein. • Electron Transfer are Edman degradation and mass spectrometry. Although Goal Dissociation (ETD) the traditional Edman technique is very robust and provides de novo capabilities, the method suffers from several • To apply electron transfer dissociation (ETD) mass • N-terminal limitations, namely throughput, sensitivity, cost, and the spectrometry to top-down protein sequencing • Protein need for specific data interpretation expertise. Mass • To apply and optimize such an approach for protein Sequencing spectrometry-based methods use the bottom-up approach, N- and C-terminal sequencing which is not only labor intensive but also has limitations •Top-down in terms of digestion efficiency and efficient capture of the Experimental N-terminal peptides. In contrast, emerging top-down approaches involve direct analysis of intact proteins. Direct Sample Preparation analysis can preserve the post-translationally modified Standard proteins were purchased from Sigma. Partially forms of proteins, resolve protein-level variations, and purified DAF-16 protein fragment was a gift from NIBS, determine expression ratios of intact protein forms.